Proof of Gene Expression During Bioaugmentation
Goal
The overall objective of this work is to develop tools for the evaluation of gene expression in microbial communities. In this proposal,
we investigate gene expression for the bioremediation of carbon tetrachloride (CT) by Pseudomonas sp. strain KC.
Rationale
Experimental justification for bioaugmentation is typically obtained by comparing the bioremediation of inoculated and uninoculated
samples. This approach is adequate for bench-scale studies. At full scale, however, design and operation of uninoculated controls is difficult
and expensive. Inadvertent inoculation of "uninoculated" regions must be avoided, and the inoculated and uninoculated regions must initially
be geologically, chemically, and biologically similar. Other methods, besides the use of uninoculated control regions, are needed to establish
that added organisms are in fact mediating the desired transformations. A logical approach is to prove expression of the genes required for the
desired transformation. Gene expression occurs at different levels as the synthesis of mRNA (transcription), the formation of polypeptides
(translation), and the biochemical reaction itself. Proof of gene expression is best obtained at each level, because each piece of evidence
strengthens the conclusion that gene expression is occurring as intended.
Approach
Transcription and translation have traditionally been established by gel electrophoresis, using Northern blots for mRNA and
Western blots and 2-D gels for proteins. These methods are not suitable for microbial communities containing large numbers of diverse
biomolecules because they do not provide sufficient discrimination and sensitivity. In this proposal, we focus on the use and development of
new tools to permit analysis of gene expression within microbial communities. The tools to be evaluated are cDNA microarrays for mRNA
transcripts and Surface Enhanced Laser Desorption Ionization Mass Spectrometry (SELDI-MS) for proteins. We will focus on Pseudomonas
sp. strain KC and on CT transformation by this strain. To identify mRNA transcripts and proteins for the genes that encode CT
transformation, we will use mutants that we have previously generated and characterized. We will then search for diagnostic mRNA transcripts
and proteins in mixtures of strain KC and Schoolcraft aquifer flora. The proposed research will pioneer testing and development of both
microarray technology and SELDI-MS as diagnostic tools for environmental applications.
Status
This project was just funded, and we are now initiating work.
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Last modified on: March 16, 2000.
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